Abstract
AimWe report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots.BackgroundVIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function.ResultsWe developed an African cassava mosaic virus—based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots.ConclusionsThe African cassava mosaic virus—based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.
Highlights
virus-induced gene silencing (VIGS) is a powerful tool to trigger transient sequence-specific gene silencing in planta
The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots
Because agro-inoculation with the VIGS-multiple cloning site (MCS) and VIGS-Chl1 agroclones using the LBA4404 strain did not result in symptomatic cassava
Summary
VIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Reverse genetics tools such as virus-induced gene silencing (VIGS) are Cassava (Manihot esculenta Crantz) is one of the most important root crops worldwide, producing starchy roots used as staple food by more than 800 million people [6]. Efficient reverse genetic tools appear as suitable to investigate the function of cassava genes for example those identified in large-scale omics studies [13,14,15]. Those studies are facilitated by the release of cassava reference genome sequences [16,17,18]
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